CDR-grafted anti-tissue factor antibodies and methods of uses there of

ABSTRACT

The present invention provides CDR-grated antibodies against human tissue factor that retain the high binding affinity of rodent monoclonal antibodies against tissue factor but have reduced immunogenicity. The present humanized antibodies are potent anticoagulants and are thus useful in the treatment and prophylaxis of human thrombotic disease. The invention also provides methods of making the CFR-grafted antibodies and pharmaceutical compositions for the attenuation or prevention of coagulation.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/313,392, filed Dec. 4, 2002, now granted under U.S. Pat No.7,235,380, which is a continuation of U.S. application Ser. No.08/480,120 filed Jun. 7, 1995, now abandoned, both of which are entirelyincorporated herein by reference.

FIELD OF THE INVENTION

Monoclonal antibodies capable of inhibiting tissue factor (TF) areuseful as anticoagulants. Conventional rodent monoclonal antibodies,however, have limited use in human therapeutic and diagnosticapplications due to immunogenicity and short serum halflife. The presentinvention provides CDR-grafted monoclonal antibodies against TF thatretain the high binding affinity of rodent antibodies but have reducedimmunogenicity. The present humanized antibodies are potentanticoagulants and are thus useful in the treatment and prophylaxis ofhuman thrombotic disease. The invention also provides methods of makingthe CDR-grafted antibodies and pharmaceutical compositions for theattenuation or prevention of coagulation.

BACKGROUND OF THE INVENTION

The coagulation of blood involves a cascading series of reactionsleading to the formation of fibrin. The coagulation cascade consists oftwo overlapping pathways, both of which are required for hemostasis. Theintrinsic pathway comprises protein factors present in circulatingblood, while the extrinsic pathway requires tissue factor, which isexpressed on the cell surface of a variety of tissues in response tovascular injury . Davie et al., 1991, Biochemistry 30: 10363. Agentsthat interfere with the coagulation cascade, such as heparin andcoumarin derivatives, have well-known therapeutic uses in theprophylaxis of venous thrombosis. Goodman and Gilman, eds., 1980, ThePharmacological Basis of Therapeutics, MacMillan Publishing Co., Inc.,New York.

Tissue factor (TF) has been investigated as a target for anticoagulanttherapy. TF is a membrane glycoprotein that functions as a receptor forfactor VII and VIIa and thereby initiates the extrinsic pathway of thecoagulation cascade in response to vascular injury. In addition to itsrole in the maintenance of hemostasis by initiation of blood clotting,TF has been implicated in pathogenic conditions. Specifically, thesynthesis and cell surface expression of TF has been implicated invascular disease (Wilcox et al., 1989, Proc. Natl. Acad. Sci. 86:2839and gram-negative septic shock (Warr et al., 1990, Blood 22:1481).

Ruf et al, (1991), Thrombosis and Haemostasis-:529) characterized theanticoagulant potential of murine monoclonal antibodies against humanTF. The inhibition of TF function by most of the monoclonal antibodiesthat were assessed was dependent upon the dissociation of the TF!Vllacomplex that is rapidly formed when TF contacts plasma. Such antibodieswere thus relatively slow inhibitors of TF in plasma. One monoclonalantibody, TF8-5G9, was capable of inhibiting the TF!Vlla complex withoutdissociation of the complex, thus providing an immediate anticoagulanteffect in plasma. Ruf et al. suggest that mechanisms that inactivate theTF!Vlla complex, rather than prevent its formation, may providestrategies for interruption of coagulation in vivo.

The therapeutic use of monoclonal antibodies against TF is limited inthat currently available monoclonals are of rodent origin. The use ofrodent antibodies in human therapy presents numerous problems, the mostsignificant of which is immunogenicity. Repeated doses of rodentmonoclonal antibodies have been found to elicit an antibody (HAMA),which can result in immune complex disease and/or neutralization of thetherapeutic antibody. See, e.g., Jaffers et al (1986) Transplantation11:572. While the use of human monoclonal antibodies would address thislimitation, it has proven difficult to generate large amounts of humanmonoclonal antibodies by conventional hybridoma technology.

Recombinant technology has been used in an effort to construct“humanized” antibodies that maintain the high binding affinity of rodentmonoclonal antibodies but exhibit reduced immunogenicity in humans.Chimeric antibodies have been produced in which the variable (V) regionof a mouse antibody is combined with the constant (c) region of a humanantibody in an effort to maintain the specificity and affinity of therodent antibody but reduce the amount of protein that is nonhuman andthus immunogenic. While the immune response to chimeric antibodies isgenerally reduced relative to the corresponding rodent antibody, theimmune response cannot be completely eliminated, because the mouse Vregion is capable of eliciting an immune response. Lobuglio et al (1989)Proc. Natl. Acad. Sci 86:4220; Jaffers et al (1986) Transplantation41:572.

In a recent approach to reducing immunogenicity of rodent antibodies,only the rodent complementarity determining regions (CDRs), rather thanthe entire V doman, are transplanted to a human antibody. Such humanizedantibodies are known as CDR grafted antibodies. CDRs are regions ofhypervariability in the V regions that are flanked by relativelyconserved regions known as the framework (FR) regions. Each V domaincontains three CDRs flanked by four FRs. The CDRs fold to form theantigen binding site of the antibody, while the FRs support thestructural conformations of the V domains. Thus by transplanting therodent CDRs to a human antibody, the antigen binding domain cantheoretically also be transferred. Owens et al (1994) J. Immunol.Methods 168:149 and Winter et al (1993) Immunology Today 11:243 reviewthe development of CDR-grafted antibodies.

Orlandi et al (1989) Proc. Natl. Acad. Sci USA 86:3833 constructed ahumanized antibody against the relatively simple hapten nitrophenacetyl(NP). The CDRgrafted antibody contained mouse CDRs and human FRs, andexhibited NP binding activity similar to the native mouse antibody.However, the construction of CDR grafted antibodies recognizing morecomplex antigens has resulted in antibodies having binding activitysignificantly lower than the native rodent antibodies.

In numerous cases it has been demonstrated that the mere introduction ofrodent CDRs into a human antibody background is insufficient to maintainfull binding activity, perhaps due to distortion of the CDR conformationby the human FR.

For example, Gorman et al (1991) Proc. Natl. Acad. Sci. 88:4181 comparedtwo humanized antibodies against human CO4 and observed considerablydifferent avidities depending upon the particular human framework regionof the humanized antibody. Co et al. Proc. Natl. Acad. Sci. USA 88:2869required a refined computer model of the murine antibody of interest inorder to identify critical amino acids to be considered in the design ofa humanized antibody. Kettleborough et al (1991) Protein Engineering1:773 report the influence of particular FR residues of a CDR-graftedantibody on antigen binding, and propose that the residues may directlyinteract with antigen, or may alter the conformation of the CDR loops.Similarly, Singer et al (1993) J. Immunol. 150:2844 report that optimalhumanization of an anti-CO18 murine monoclonal antibody is dependentupon the ability of the selected FR to support the CDR in theappropriate antigen binding conformation. Accordingly, recreation of theantigen binding site requires consideration of the potential intrachaininteractions between the FR and CDR, and manipulation of amino acidresidues of the FR that maintain contacts with the loops formed by theCDRs. While general theoretical guidelines have been proposed for thedesign of humanized antibodies (see e.g., Owens et al), in all cases theprocedure must be tailored and optimized for the particular rodentantibody of interest.

There is a need in the art for humanized antibodies with reducedimmunogenicity and comparable binding affinity relative to the parentrodent antibody for various therapeutic applications. In particular,there is a need for a humanized antibody against human tissue factorhaving anticoagulant activity and useful in the treatment and preventionof thrombotic disease.

SUMMARY OF THE INVENTION

The present invention is directed to CDR grafted antibodies capable ofinhibiting human tissue factor wherein the CDRs are derived from anon-human monoclonal antibody against tissue factor and the FR andconstant (c) regions are derived from one or more human antibodies. In apreferred embodiment, the murine monoclonal antibody is TF8-5G9.

In another embodiment, the present invention provides a method ofproducing a CDR-grafted antibody capable of inhibiting human tissuefactor which method comprises constructing one or more expressionvectors containing nucleic acids encoding CDR-grafted antibody heavy andlight chains, transfecting suitable host cells with the expressionvector or vectors, culturing the transfected host cells, and recoveringthe CDR-grafted antibody.

The present invention also provides a method of attenuation ofcoagulation comprising administering a CDR-grafted antibody capable ofinhibiting human tissue factor to a patient in need of such attenuation.

The present invention further provides a method of treatment orprevention of thrombotic disease comprising administering a CDR-graftedantibody capable of inhibiting human tissue factor to a patient in needof such treatment or prevention. In a preferred embodiment, thethrombotic disease is intravascular coagulation, arterial restenosis orarteriosclerosis.

Another embodiment of the present invention is directed to apharmaceutical composition comprising CDR grafted antibodies capable ofinhibiting human tissue factor and further comprising a pharmaceuticallyacceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A through 1E provide the nucleotide and deduced amino acidsequences of the heavy chain of murine monoclonal antibody TF8-SG9.

FIGS. 2A through 2C provide the nucleotide and deduced amino acidsequences of the light chain of murine monoclonal antibody TF8-5G9.

FIG. 3 is a graph depicting the ability of CDR-grafted antibodyTF8HCDR1×TF8LCDR1 to bind to human tissue factor and to compete withmurine monoclonal antibody TF85G9 for binding to tissue factor. Solidsymbols indicate direct binding of TF8HCDR1×TF8LCDR1 and the positivecontrol chimeric TF85G9 to tissue factor. Open symbols indicatecompetition binding of TF8HCDR1×TF8LCDR1 or chimeric TF85G9 with murinemonoclonal antibody TF85G9.

FIGS. 4A through 4S present the DNA sequence of expression vectorpEe6TF8HCDR20 (SEQ ID NO: 15) and the amino acid sequence of the codingregion 11 of the CDR-grafted heavy chain TF8HCDR20 (SEQ ID NOS:16, 17,18, and 19).

FIGS. 5A through 5U present the DNA sequence of expression vectorpEe12TF8LCDR3 and the amino acid sequence (SEQ ID NO:20) of the codingregions of the CDR-grafted light chain TF8LCDR3.

FIG. 6 is a graph depicting the ability of CDR-grafted antibodyTF8HCDR20×TF8LCDR3 to bind to human tissue factor.

FIG. 7 is a graph depicting the ability of CDR-grafted antibodyTF8HCDR20×TF8LCDR3 to compete with murine monoclonal antibody TF85G9 forbinding to tissue factor.

FIG. 8 is a graph depicting the ability of CDR-grafted antibodyTF8HCDR20×TF8LCDR3 to inhibit factor X activation.

FIG. 9 provides expression vector pEe6TF8HCDR20 resulting from thesubcloning of CDR grafted heavy chain TF8HCDR20 into myeloma expressionvector pEehCMV-Bg1I. The following abbreviations are used: VH is theCDR-grafted heavy chain variable region; Cy4 is the human IgG4 constantregion; pA is the polyadenylation signal; ampR is the B-lactamase gene;and hCMV is human cytomegalovirus.

FIG. 10 provides expression vector pEe12TF8LCRD3 resulting from thesubcloning of CDR grafted light chain TF8LCDR3 into myeloma expressionvector pEe12. The following abbreviations are used: VL is theCDR-grafted light chain variable region; CK is the human kappa constantregion; SVE is the SV40 early promoter; GS is glutamine synthetase cDNA.Other abbreviations are as noted in FIG. 9.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides CDR- grafted antibodies capable ofinhibiting human tissue factor wherein the CDRs are derived from anon-human monoclonal antibody against tissue factor and the FR and Cregions are derived from one or more human antibodies. The presentinvention further provides methods of making and using the subjectCDR-grafted antibodies.

In accordance with the present invention, the CDR-grafted antibody is anantibody in which the CDRs are derived from a non-human antibody capableof binding to and inhibiting the function of human tissue factor, andthe FR and C regions of the antibody are derived from one or more humanantibodies. The CDRs derived from the non-human antibody preferably haveabout 90% to about 100% identity with the CDRs of the nonhuman antibody,although any and all modifications, including substitutions, insertionsand deletions, are contemplated so long as the CDR-grafted antibodymaintains the ability to bind to and inhibit tissue factor. The regionsof the CDR-grafted antibodies that are derived from human antibodiesneed not have 100% identity with the human antibodies. In a preferredembodiment, as many of the human amino acid residues as possible areretained in order than immunogenicity is negligible, but the humanresidues, in particular residues of the FR region, are substituted asrequired and as taught herein below in accordance with the presentinvention. Such modifications as disclosed herein are necessary tosupport the antigen binding site formed by the CDRs while simultaneouslymaximizing the humanization of the antibody.

Non-human monoclonal antibodies against human tissue factor from whichthe CDRs can be derived are known in the art (Ruf et al., 1991; Morriseyet al., 1988, Thrombosis Research 52:247) or can be produced bywell-known methods of monoclonal antibody production (see e.g. Harlow etal., eds., 1988, Antibodies A Laboratory Manual, Cold Spring HarborLaboratories, Cold Spring Harbor, N.Y.). Purified human tissue factoragainst which monoclonal antibodies can be raised is similarlywell-known (Morrisey et al., 1987, Cell 50:129) and available to theskilled artisan. Murine monoclonal antibodies, and in particular murinemonoclonal antibody TF8-5G9 disclosed by Ruf et al. and Morrisey et al.,1988, Thrombosis Research 52:247, and U.S. Pat. No. 5,223,427 areparticularly preferred.

The ordinarily skilled artisan can determine the sequences of the CDRsby reference to published scientific literature or sequence databanks,or by cloning and sequencing the heavy and light chains of theantibodies by conventional methodology. In accordance with the presentinvention, the cDNA and amino acid sequences of the heavy chain (SEQ IDNOS: 1 and 2, respectively) and light chain (SEQ ID NOS.: 3 and 4,respectively) of murine monoclonal antibody TF8-5G9 are provided. ThecDNA and deduced amino acid sequence of the murine TF8-5G9 are providedin FIGS. 1A through 1E. The cDNA and deduced amino acid sequence of themurine TF8-5G9 light chain is provided in FIGS. 2A through 2C.

Each of the heavy and light chain variable regions contain three CDRsthat combine to form the antigen binding site. The three CDRs aresurrounded by four FR regions that primarily function to support theCDRs. The sequences of the CDRs within the sequences of the variableregions of the heavy and light chains can be identified bycomputer-assisted alignment according to Kabat et al (1987) in Sequencesof Proteins of Immunological Interest 4^(th) ed., United StatesDepartment of Health and Human Services, US Government Printing Office,Washington, D.C., or by molecular modeling of the variable regions, forexample utilizing the ENCAD program as described by Levitt (1983) J.Mol. Biol. 168:595.

In a preferred embodiment the CDRs are derived from murine monoclonalantibody TF8-5G9. The preferred heavy chain CDRs have the followingsequences:

CDR1 DYYMH (SEQ ID NO:5) CDR2 LIDPENGNTIYDPKFQG (SEQ ID NO:6) CDR3DNSYYFDY (SEQ ID NO:7)The preferred light chain CDRs have the following sequences:

CDR1 KASQDIRKYLN (SEQ ID NO:8) CDR2 YATSLAD (SEQ ID NO:9) CDR3 LQHGESPYT(SEQ ID NO:10)The sequences of the CDRs of the murine or other non-human antibody, andin particular the sequences of the CDRs of the TF8-5G9, may be modifiedby insertions, substitutions and deletions to the extent that the CDRgrafted antibody maintains the ability to bind to and inhibit humantissue factor. The ordinarily skilled artisan can ascertain themaintenance of this activity by performing the functional assaysdescribed herein below. The CDRs can have, for example, from about 50%to about 100% homology to the CDRs of SEQ ID NOS: 5-10. In a preferredembodiment the CDRs have from about 80% to about 100% homology to theCDRs of SEQ ID NOS: 5-10. In a more preferred embodiment the CDRs havefrom about 90% to about 100% homology to the CDRs of the SEQ IDNOS.5-10.

The FR and C regions of the CDR-grafted antibodies of the presentinvention are derived from one or more human antibodies. Humanantibodies of the same class and type as the antibody from which theCDRs are derived are preferred. The FR of the variable region of theheavy chain is preferably derived from the human antibody KOL (Schmidtet al., 1983, Hoppe-Seyler's Z. Physiol. Chern. 364:713. The FR of thevariable region of the light chain is preferably derived from the humanantibody REI. (EPP et al 1974. Eur. J. Biochem. 45:513). In accordancewith the present invention, it has been discovered that certain residuesof the human FR are preferably replaced by the corresponding residue ofthe non-human antibody from which the CDRs are derived. For example,certain FR residues of the TF8-5G9 are preferably retained to achieveoptimal binding to antigen.

For convenience, the numbering scheme of Kabat et al has been adoptedherein. Residues are designated by lower case numbers of hyphens asnecessary to conform the present sequences to the standard Kabatnumbered sequence.

In accordance with the present invention, residues that are retained inthe FR region, e.g. residues that are not replaced by human FR residues,are determined according to the following guidelines. Residues that areidiosyncratic to the parent antibody, e.g. TF8-5G9, relative to a humanconsensus sequence of Kabat et al, are retained. Residues of the parentantibody that are in agreement with the consensus sequence are retainedif the corresponding residue of the human antibody e.g. KOL or REI, isidiosyncratic. Residues that are part of the antibody loop canonicalstructures defined by Chothia et al (1989) Nature 342:877, such asresidue 71 of the heavy and light chains, are retained. FR residuespredicted to form loops, such as residues 28-30 of the heavy chain, areretained. FR residues predicted to influence the conformation of theCDRs such as residues 48 and 49 preceding CDR2 of the heavy chain, areretained. Residues that have been demonstrated to be critical in thehumanization of other antibodies may also be retained. The foregoingguidelines are followed to the extent necessary to support the antigenbinding site formed by the CDRs while simultaneously maximizing thehumanization of the antibody.

The amino acid sequence of a representative CDR-grafted heavy chainvariable region derived from murine monoclonal antibody TF8-SG9 andhuman antibody KOL is shown below. The CDR-grafted heavy chain isdesignated TF8HCDR1; murine residues were retained in the FR at residues6, 17, 23, 24, 28, 29, 30, 48, 49, 68, 71, 73, 78, 88 and 91. CDRs areunderlined.

         10        20       30       35ab            50 (SEQ ID NO:11)QVQLVQSGGG VVQPGRLLRL SCKASGFNIK DYYMH--WVR QAPGKGLEWIG52abc    60           70            80 82abc      90LIDP--ENGNTIYD PKFQGRFSIS ADTSK--NTAFL QMDSLRPEDTAVY 100        110YCARDNSYYF DYWGQGTPVT VSS

The amino acid sequence of a representative CDR-grafted light chainvariable region derived from murine monoclonal antibody TF8-5G9 andhuman antibody REI is shown below. The CDR-grafted light chain isdesignated TF8LCDR1; murine residues were retained in the FR at residues39, 41, 46 and 105. CDRs are underlined.

        10         20         30        40         50 (SEQ ID NO:12)DIQMTQSPSS LSASVGDRVT ITCKASQDIR KYLNWYQQK WKAPKTLIYY        60         70         80         90        100 ATSLADGVPSRFSGSGSGTD YTFTISSLQP EDIATYYCLQ HGESPYTFGQ GTKLEITR

A CDR-grafted antibody containing variable regions TF8HCDR1 and TF8LCDR1has been demonstrated in accordance with the present invention to be aseffective as murine monoclonal antibody TF8-5G9 in binding to humantissue factor. It has been further discovered in accordance with thepresent invention, by examination of the molecular structure of murinemonoclonal antibody TF8-5G9, and by design, construction, and analysisof CDR-grafted antibodies, that the FR regions can be further humanizedwithout the loss of antigen binding activity. In particular, the FRregion may retain the human FR residue at residues 6, 17, 68, 73 and 78of the heavy chain and residues 39, 41, 16 and 105 of the light chain,with maintenance of antigen binding activity.

In a most preferred embodiment, the heavy chain variable region containsa FR derived from human antibody KOL in which murine monoclonal antibodyTF8-5G9 residues are retained at amino acids 23, 24, 28, 29, 30, 48, 49,71, 88 and 91. The preferred heavy chain variable region is designedTF8HCDR20 and has the following sequence.

        10         20         30    35ab             50 (SEQ ID NO:13)QVQLVESGGG VVQPGRSLRL SCKASGFNIK DYYMH--WVR QAPGKGLEWIGL52abc      60         70        80  82abc      90        100IDP--ENGNTIYD PKFQGRFTIS ADNSKNTLFL QMDSLRPEDTAVY YCARDNSYYF        110DYWGQGTPVT VSS

In a most preferred embodiment, the light chain variable region containsa FR derived from human antibody REI in which murine monoclonal antibodyTF8-5G9 residues are retained at amino acids 39 and 105. The preferredlight chain variable region is designated TF8LCDR20 and has thefollowing sequence.

        10         20         30         40         50 (SEQ ID NO:14)DIQMTQSPSS LSASVGDRVT ITCKASQDIR KYLNWYQQKP GKAPKLLIYY        60         70         80         90        100 ATSLADGVPSRFSGSGSGTD YTFTISSLQP EDIATYYCLQ HGESPYTFGQ GTKLEITR

It is within the ken of the ordinarily skilled artisan to make minormodifications of the foregoing sequences, including amino acidsubstitutions, deletions and insertions. Any such modifications arewithin the scope of the present invention so long as the resultingCDR-grafted antibody maintains the ability to bind to and inhibit humantissue factor. The ordinarily skilled artisan can assess the activity ofthe CDR-grafted antibody with reference to the functional assaysdescribed herein below.

The human constant region of the CDR-grafted antibodies of the presentinvention is selected to minimize effector function. The intended use ofthe CDR-grafted antibodies of the present invention is to block thecoagulation cascade by inhibition of tissue factor, and thus antibodyeffector functions such as fixation of complement are not desirable.Antibodies with minimal effector functions include IgG2, IgG4, IgA, IgDand IgE. In a preferred embodiment of the present invention, the heavychain constant region is the human IgG4 kappa constant region; and thelight chain constant region is the human IgG4 kappa constant region.

In that effector functions may not be desirable for therapeutic uses,the present invention further contemplates active fragments of theCDR-grafted antibodies, and in particular Fab fragments and F(ab′)2fragments. Active fragments are those fragments capable of inhibitinghuman tissue factor. Fab fragments and F (ab′)₂ fragments may beobtained by conventional means/for example by cleavage of theCDR-grafted antibodies of the invention with an appropriate proteolyticenzyme such as papain or pepsin, or by recombinant production. Theactive fragments maintain the antigen binding sites of the CDR-graftedantibodies and thus are similarly useful therapeutically.

The ability of the CDR-grafted antibodies designed and constructed astaught in accordance with the present invention to bind and inhibithuman tissue factor can be assessed by functional assays. For example,in a rapid and convenient assay, expression vectors containing nucleicacids encoding the CDR grafted heavy and light chains can beco-transfected into suitable host cells and transiently expressed. Theresulting antibodies can be assessed by standard assays for ability tobind human tissue factor, and for ability to compete for binding totissue factor with the human antibody from which the CDRs are derived.

For example, transient expression of nucleic acids encoding theCDR-grafted heavy and light chains in COS cells provides a rapid andconvenient system to test antibody gene expression and function. Nucleicacids encoding the CDR-grafted heavy and light chains, respectively, arecloned into a mammalian cell expression vector, for example pSG5,described by Green et al (1988) Nucleic Acids Res. 16:369 andcommercially available from Stratagene Cloning Systems, La Jolla, Calif.The pSG5 expression vector provides unique restriction sites for theinsertion of the heavy and light chain genes, and in vivo expression isunder the control of the SV40 early promoter. Transcriptionaltermination is signaled by the SV40 polyadenylation signal sequence.

The pSG5 based expression vector containing nucleic acids encoding theheavy and light chains are cotransfected into COS cells and culturedunder conditions suitable for transient expression. Cell culture mediais then harvested and examined for antibody expression, for example byan enzyme linked immunosorbent assay (ELISA), to determine that suitablelevels of antibody have been produced. An ELISA may then be used toassess the ability of the CDR-grafted antibody to bind to human tissuefactor. Human tissue factor is immobilized on a microtiter plate and theCOS cell supernatant containing the CDR-grafted antibody is addedfollowed by an incubation at room temperature for about one hour. Theplates are then washed with a suitable detergent-containing buffer suchas phosphate buffered saline (PBS)/Tween, followed by the addition ofthe components of a suitable detection system. For example, horseradishperoxidase conjugated goat antihuman kappa chain polyclonal antibody isadded, followed by washing, followed by addition of substrate forhorseradish peroxidase, and detection. The CDR-grafted antibodies withinthe scope of the present invention are those which are capable ofbinding to human tissue factor to a degree comparable to the non-humanantibody from which the CDRs are derived as determined by the foregoingassay.

The ability of the CDR-grafted antibodies to inhibit the activity ofhuman tissue factor in vivo can be conveniently assessed by thefollowing in vitro assay that mimics in vivo coagulation events. Inresponse to vascular injury in vivo, tissue factor binds to factor VIIand facilitates the conversion of factor VII to a serine protease(factor VIla). The factor VIa-tissue factor complex converts factor X toa serine protease (factor Xa). Factor Xa forms a complex with factor Va(from the intrinsic coagulation pathway), resulting in the conversion ofprothrombin to thrombin, which in turn results in the conversion offibrinogen to fibrin. In a convenient in vitro functional assay, tissuefactor is incubated in the presence of factor VIIa and the CDR graftedanti-tissue factor antibody produced in the transient expression systemdescribed above. Factor X is added and the reaction mixture isincubated, followed by an assay for factor Xa activity utilizing achromogenic substrate for factor Xa (Spectrozyme Fxa, AmericanDiagnostica, Inc., Greenwich, Conn.). The ability of the CDR-graftedantibody to inhibit factor X activation thus provides a measure of theability of the CDR-grafted antibody to inhibit the activity of humantissue factor.

The CDR-grafted antibodies within the scope of the present invention arethose which are capable of inhibiting human tissue factor to a degreecomparable to the non-human antibody from which the CDRs are derived asdetermined by the foregoing assay. In one embodiment, the CDR-graftedantibody has at least 50% of the inhibitory activity of TF8-5G9 forhuman tissue factor. In a preferred embodiment, the CDR-grafted antibodyhas at least 70% of the inhibitory activity of TF8-5G9 for human tissuefactor. In a more preferred embodiment, the CDR-grafted antibody has atleast 80% of the inhibitory activity of TF8-5G9 for human tissue factor.In a most preferred embodiment, the CDR-grafted antibody has at least90% of the inhibitory activity of TF8-5G9 human tissue factor.

In another embodiment, the present invention provides a method ofproducing a CDR-grafted antibody capable of inhibiting human tissuefactor. The method comprises constructing an expression vectorcontaining a nucleic acid encoding the CDR-grafted antibody heavy chainand an expression vector containing a nucleic acid encoding theCDR-grafted antibody light chain, transfecting suitable host cells withthe expression vectors, culturing the transfected host cells underconditions suitable for the expression of the heavy and light chains,and recovering the CDR-grafted antibody. Alternately, one expressionvector containing nucleic acids encoding the heavy and light chains maybe utilized.

Standard molecular biological techniques, for example as disclosed bySambrook et al. (1989), Molecular Cloning: A Laboratory Manual ColdSpring Harbor Press, Cold Spring Harbor, N.Y. may be used to obtainnucleic acids encoding the heavy and light chains of the CDR-graftedantibodies of the present invention. A nucleic acid encoding theCDR-grafted variable domain may be constructed by isolating cDNAencoding the antibody to be humanized, e.g. murine monoclonal antibodyTF8-5G9, by conventional cloning methodology from the hybridomaproducing the antibody, or by polymerase chain reaction (PCR)amplification of the variable region genes, as described for example byWinter et al., followed by site-directed mutagenesis to substitutenucleotides encoding the desired human residues into the FR regions.Alternately, the cDNA encoding the human antibody can be isolated,followed by site-directed mutagenesis to substitute nucleotides encodingthe desired murine residues into the CDRs.

Nucleic acids encoding the CDR-grafted variable domain may also besynthesized by assembling synthetic oligonucleotides, for exampleutilizing DNA polymerase and DNA ligase. The resulting syntheticvariable regions may then be amplified by PCR. Nucleic acids encodingCDR-grafted variable domains may also be constructed by PCR strandoverlap methods that are known in the art and reviewed by Owens et al.

Accordingly, having determined the desired amino acid sequences of theCDR-grafted variable domains in accordance with the present invention,the ordinarily skilled artisan can obtain nucleic acids encoding thevariable domains. Further, the skilled artisan is aware that due to thedegeneracy of the genetic code, various nucleic acid sequences can beconstructed that encode the CDR-grafted variable domains. All suchnucleic acid sequences are contemplated by the present invention.

The nucleic acids encoding the CDR-grafted variable domains are linkedto appropriate nucleic acids encoding the human antibody heavy or lightchain constant region. Nucleic acid sequences encoding human heavy andlight chain constant regions are known in the art. It is within the kenof the ordinarily skilled artisan to include sequences that facilitatetranscription, translation and secretion, for example start codons,leader sequences, the Kozak consensus sequence (Kozak, 1987, J. Mol.Biol. 196:947) and the like, as well as restriction endonuclease sitesto facilitate cloning into expression vectors.

The present invention thus further provides nucleic acids encoding theheavy and light chains of CDR-grafted antibodies capable of inhibitinghuman tissue factor wherein the CDRs are derived from a murinemonoclonal antibody against tissue factor and the FR and C regions arederived from one or more human antibodies.

In accordance with the present invention, representative nucleic acidsencoding CDR-grafted heavy and light chains are constructed. TheCDR-grafted heavy chain comprises a variable region containing FRregions derived from human antibody KOL and CDRs derived from murinemonoclonal antibody TF8-5G9 and further comprises a constant regionderived from the heavy chain of human IgG4. The CDR-grafted light chaincomprises a variable region containing FR regions derived from humanantibody REI and CDRs derived from murine monoclonal antibody TF8-SG9and further comprises a constant region derived from human IgG4 kappachain. Nucleic acids encoding the heavy and light chains wereconstructed by assembling the variable regions from syntheticnucleotides, amplifying the assembled variable regions by PCR, purifyingthe amplified nucleic acids, and ligating the nucleic acid encoding thevariable region into a vector containing a nucleic acid encoding theappropriate human constant region.

The sequences of representative nucleic acids encoding CDR-grafted heavyand light chains are presented as nucleotides 1-2360 of SEQ ID No. 15and nucleotides 1-759 of SEQ ID NO:20, respectively.

The nucleic acid sequence encoding a preferred heavy chain (nucleotides1-2360 of SEQ ID NO: 15) is designated the TF8HCDR20 gene. The nucleicacid sequence contains the following regions: 5′ EcoRI restriction site(nucleotides 1-6); Kozak sequence (nucleotides 7-15); start codon andleader sequence (nucleotides 16-72); CDR-grafted variable region(nucleotides 73-423); human IgG4 CHI domain (nucleotides 424-717); humanIgG4 intron 2 (nucleotides 718-1110); human IgG4 hinge (nucleotides1111-1146); human IgG4 intron 3 (nucleotides 1147-1267); human IgG4 CH2domain (nucleotides 1268-1594); human IgG4 intron 4 (nucleotides1595-1691); human IgG4 CH3 domain (nucleotides 1692-2012); 3′untranslated region (nucleotides 2013-2354); 3′ BamHI end spliced toBc1I site of expression vector (nucleotides 2355-2360).

The nucleic acid sequence encoding a preferred light chain gene(nucleotides 1-759 of SEQ ID NO:20) is designated the TF8LCDR3 gene. Thenucleic acid sequence contains the following regions: 5; EcoRIrestriction site (nucleotides 1-5); Kozak sequence (nucleotides 6-8);start codon and leader sequence (nucleotides 9-68); CDR-grafted variableregion (nucleotides 69-392); human kappa constant region (nucleotides393-710); 3 ′ untranslated region (nucleotides 711-753); 3′ BamHI endspliced to Bcll site of expression vector (nucleotides 754-759).

The foregoing preferred sequences can be modified by the ordinarilyskilled artisan to take into account degeneracy of the genetic code, andto make additions, deletions, and conservative and nonconservativesubstitutions that result in a maintenance of the function of thenucleic acid, i.e. that it encodes a heavy or light chain of aCDR-grafted antibody capable of inhibiting human tissue factor.Restriction sites and sequences that facilitate transcription andtranslation may be altered or substituted as necessary depending uponthe vector and host system chosen for expression.

Suitable expression and hosts for production of the CDR-graftedantibodies of the present invention are known to the ordinarily skilledartisan. The expression vectors contain regulatory sequences, such asreplicons and promoters, capable of directing replication and expressionof heterologous nucleic acids sequences in a particular host cell. Thevectors may also contain selection genes, enhancers, signal sequences,ribosome binding sites, RNA splice sites, polyadenylation sites,transcriptional terminator sequences, and so on. The vectors may beconstructed by conventional methods well-known in the art, or obtainedfrom commercial sources. The expression vectors preferably haveconvenient restriction sites at which the nucleic acids encoding theantibody chains of the invention are inserted. Myeloma expressionvectors in which antibody gene expression is driven by the humancytomegalovirus promoter-enhancer or are particularly preferred.

Expression vectors containing a nucleic acid encoding the CDR-graftedheavy chain under the control of a suitable promoter and expressionvectors containing a nucleic acid encoding the CDR-grafted light chainunder the control of a suitable promoter are cotransfected into asuitable host cell. In another embodiment, nucleic acids encoding bothheavy and light chains are provided in a single vector for transfectionof a suitable host cell.

Suitable host cells or cell lines for expression of the CDR-graftedantibodies of the present invention include bacterial cells, yeastcells, insect cells, and mammalian cells such as Chinese hamster ovary(CHO) cells, COS cells, fibroblast cells and myeloid cells. Mammaliancells are preferred. CHO, COS and myeloma cells are particularlypreferred. Myeloma cells are preferred for establishing permanentCDR-grafted antibody producing cell lines. Expression of antibodies inmyeloma cells, bacteria, and yeast is reviewed by Sandhu (1992) CriticalReviews in Biotechnology 12:437. Expression in mammalian cells isreviewed by Owen et al.

Transfection of host cells by the expression vectors containing nucleicacids encoding the CDR grafted heavy and light chains can beaccomplished by well-known to methods one of ordinary skill in the art.Such methods include, for example, calcium chloride transfection,calcium phosphate transfection, lipofection and electroporation.Suitable culture methods and conditions for the production of the CDRgrafted antibodies are likewise well-known in the art. The CDR-graftedantibodies can be purified by conventional methods, including ammoniumsulfate precipitation, affinity chromatography, gel electrophoresis, andthe like. The ability of the CDR grafted antibodies to bind to andinhibit human tissue factor can be assessed by the in vitro assaysdescribed above.

The CDR-grafted antibodies of the present invention have a variety ofutilities. For example, the antibodies are capable of binding to humantissue factor and thus are useful in assays for human tissue factor frombody fluid samples, purification of human tissue factor, and so on.

The CDR-grafted antibodies of the present invention are capable ofinhibiting human tissue factor. Human tissue factor is well-known to bean essential element in the human coagulation cascade. The ability ofthe antibodies of the present invention to disrupt the coagulationcascade is demonstrated by in vitro assays in which the antibodiesprevent factor X activation. Accordingly, the present antibodies areuseful in the attenuation of coagulation. The present invention thusprovides a method of attenuation of coagulation comprising administeringa therapeutically effective amount of CDR-grafted antibody capable ofinhibiting human tissue factor to a patient in need of such attenuation.

Numerous thrombotic disorders are characterized by excessive orinappropriate coagulation and are effectively treated or prevented byadministration of agents that interfere with the coagulation cascade.Accordingly, the present invention further provides a method oftreatment or prevention of a thrombotic disorder comprisingadministering a therapeutically effective amount of a CDR-graftedantibody capable of inhibiting human tissue factor to a patient in needof such treatment or prevention. In a preferred embodiment, thethrombotic disorder is intravascular coagulation, arterial restenosis orarteriosclerosis. The antibodies of the invention may be used incombination with other antibodies or therapeutic agents.

A therapeutically effective amount of the antibodies of the presentinvention can be determined by the ordinarily skilled artisan withregard to the patient's condition, the condition being treated, themethod of administration, and so on. A therapeutically effective amountis the dosage necessary to alleviate, eliminate, or prevent thethrombotic disorder as assessed by conventional parameters. For example,a therapeutically effective dose of a CDR-grafted antibody of thepresent invention may be from about 0.1 mg to about 20 mg per 70 kg ofbody weight. A preferred dosage is about 1.0 mg to about 5 mg per 70 kgof body weight.

A patient in need of such treatment is a patient suffering from adisorder characterized by inappropriate or excessive coagulation, or apatient at risk of such a disorder. For example, anticoagulant therapyis useful to prevent postoperative venous thrombosis, and arterialrestenosis following balloon angioplasty.

The CDR-grafted antibodies of the present invention are useful in thesame manner as comparable therapeutic agents, and the dosage level is ofthe same order of magnitude as is generally employed with thosecomparable therapeutic agents. The present antibodies may beadministered in combination with a pharmaceutically acceptable carrierby methods known to one of ordinary skill in the art.

Another embodiment of the present invention is directed to apharmaceutical composition comprising at least one CDR-grafted antibodycapable of inhibiting human tissue factor and further comprising apharmaceutically acceptable carrier. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like. The use of such media and agents forpharmaceutically active substances is well-known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

The antibodies can be administered by well-known routes including oraland parenteral, e.g., intravenous, intramuscular, intranasal,intrdermal, subcutaneous, and the like. Parenteral administration andparticularly intravenous administration is preferred. Depending on theroute of administration, the pharmaceutical composition may requireprotective coatings.

The pharmaceutical forms suitable for injectionable use include sterileaqueous solutions or dispersions and sterile powers for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the ultimate solution form must be sterile andfluid. Typical carriers include a solvent or dispersion mediumcontaining, for example, water buffered aqueous solutions (i.e.,biocompatible buffers), ethanol, polyol such as glycerol, propyleneglycol, polyethylene glycol, suitable mixtures thereof, surfactants orvegetable oils. The antibodies may be incorporated into liposomes forparenteral administration. Sterilization can be accomplished by anart-recognized techniques, including but not limited to, addition ofantibacterial or antifungal agents, for example, paraben, chlorobutanol,phenol, sorbic acid or thimersal. Further, isotonic agents such assugars or sodium chloride may be incorporated in the subjectcompositions.

Production of sterile injectable solution containing the subjectantibodies is accomplished by incorporating these antibodies in therequired amount in the appropriate solvent with various ingredientsenumerated above, as required, followed by sterilization, preferablyfilter sterilization. To obtain a sterile powder, the above solutionsare vacuum dried or freeze-dried as necessary.

The following examples further illustrate the present invention.

EXAMPLE 1 Isolation and Sequencing of TF8-5G9 Light Chain (LC) and HeavyChain (HC)

Two DNA libraries were generated from oligo (dT)-primed TF8-5G9hybridoma RNA utilizing standard molecular biology procedures asdescribed by Sambrook et al. The cDNA was cloned into the Librarian IIplasmid vector from Invitrogen (San Diego, Calif.), and the librarieswere screened for cDNA clones encoding murine IgG HC and LC. Afull-length cDNA clone for the heavy chain could not be isolated,despite the construction of two independent libraries. A random primedTF8-5G9 cDNA library was generated to obtain the missing 5′ sequence ofthe heavy chain. Consequently, the heavy chain cDNA was in two pieces: a5′ clone of 390 nucleotides and a 3′ clone of 1392 nucleotides. The twoHC clones overlap by 292 nucleotides.

The HC and LC clones were completely sequenced were completely sequencedby the dideoxy chain termination method of Sanger et al. (1977) Proc.Natl. Acad. Sci. USA 74:5463. To verify the variable region sequence,sequence was obtained from PCR-amplified cDNA that had been synthesizedfrom total TF8-5G9 hybridoma RNA. Total TF8-5G9 hybridoma RNA wasisolated by the guanidinium thiocyanate method of Chrigwin et al (1970)Biochemistry 18:5294. cDNA was synthesized using the Perkin Elmer(Norwalk, Conn.) GeneAmp RNA Polymerase Chain Reaction (PCR) kit with anoligo (dT) primer. Components of the same kit were used in the PCR toamplify the LC and HC variable regions using primers based on thesequence that had been obtained for the cDNA clones. The amplifiedvariable region fragments were gel-purified and sequenced according tothe method of Tracy et al (1991) BioTechniques 11:68 on a Model 373AApplied Biosystems, Inc. (Foster City, Calif.) automated fluorescent DNAsequencer. The sequence for TF8-5G9 LC and HC obtained from RNAamplification and the sequence obtained from the cDNA clones agreed. TheTF8-5G9 HC variable region sequence with protein translation is shown inFIG. 1 and SEQ ID NO: 1, and that for the LC is shown in FIG. 2 and SEQID NO:3.

EXAMPLE 2 Chimeric LC and HC Expression Vector Construction

In order to test the binding activity of the CDR-grafted anti-TF LC andHC individually, mouse-human chimeric TF8-5G9 LC and HC wereconstructed. This allowed the CDR-grafted LC to be tested for TF bindingability in combination with the chimeric HC, and the CDR-grafted HC tobe tested in combination with the chimeric LC.

Primers were designed to amplify the TF8-5G9 LC variable region using astemplate cDNA clones in the Librarian II vector. The 5′ primer wasdesigned with an EcoRI site while the 3′ primer was designed with a NarIsite. PCR was used to amplify the LC variable region, generating a 433bp fragment with a 5′ EcoRI end and 3′ NarI end. The fragment includedthe signal sequence from the TFB-5G9 Lc cDNA clone but incorporated a 2base change in the arginine codon immediately following the ATG startcodon. This change retained the arginine residue but made the sequenceconform to the Kozak consensus sequence in order to potentially improvetranslation of the LC mRNA. The PCR amplified LC variable regionfragment was digested with EcoRI and NarI restriction enzymes andpurified by electrophoresis on a 2% Nusieve, 1% Seakem agarose gel (FMCBio Products, Rockland, Me.).

The DNA was extracted from the gel slice and purified by the Geneclean(Bio 101, La Jolla, Calif.) procedure. The full-length chimeric TF8-5G9LC gene was generated by cloning this DNA into the EcoRI and NarI sitesof a pSP73 vector (Promega, Madison, Wis.) which contains the humankappa constant region. The gene was isolated from the pSP73 vector byEcoRI digestion and subcloned into the EcoRI site of the pSG5 mammaliancell expression vector (Stratagene Cloning Systems, La Jolla, Calif.).

The chimeric TF8-5G9 HC gene was assembled in a manner similar to thatof the chimeric LC. Since there was no full-length HC cDNA isolated fromthe Librarian II vector cDNA libraries, the HC variable region fragmentthat was generated by the PCR from total TF8-5G9 hybridoma cell RNA wasused as the template. Primers which incorporated an EcoRI site at the 5′end and a SacI site at the 3′ end were used in the PCR to generate a 430bp fragment which contained the TF8-5G9 HC Kozak sequence, start codon,signal sequence, and variable region. This fragment was digested withthe restriction enzymes EcoRI and SacI, and gel-purified using the sameprocedure that was used with the chimeric LC construction.

The full-length TF8-5G9 chimeric HC gene was constructed by cloning thevariable region fragment into the EcoRI and SacI sites of the pSG5expression vector containing the human IgG4 constant region.

EXAMPLE 3 Design and Construction of the CDR-Grafted heavy and LightChain Genes

The variable region domains of the CDR-grafted HC and LC genes weredesigned with an EcoRI overhang at the 5′ end followed by a Kozaksequence to improve antibody expression. The leader sequences werederived from the heavy and light chains of the murine monoclonalantibody B72.3 (Whittle et al. (1987) Protein Engineering 1:499). The 3′end of the variable regions were designed to have overhangs whichallowed for splicing to the appropriate human constant region DNA.

In the initially designed CDR-grafted TF8-5G9 heavy and light chains theCDRs were derived from murine TF8-5G9 sequence while the frameworks werederived primarily from human antibody sequence. The human antibody KOL(Schmidt et al.) was used for the heavy chain frameworks, while thehuman antibody dimer (EPP et al.) was used for the light chainframeworks.

Several criteria were used to select murine framework residues in thedesign of the TF8-5G9 CDR-grafted heavy and light chain variableregions. Framework residues which, at a particular position, areidiosyncratic to TF8-SG9, were retained as murine sequence with theassumption that they contributed to its unique binding characteristics.TF8-5G9 murine residues were also retained at framework positions wherethey were in agreement with the human consensus sequence but where thecorresponding residues in KOL or REI were idiosyncratic. Residues thatare part of antibody loop canonical structures such as residue 71(numbering according to Kabat et al.) of the heavy and light chains werealso retained as murine sequence. Framework residues that form loopssuch as residues 26-30 of the HC were kept as TF8-5G9 murine sequence atpositions were the murine sequence differed from the human. Residuesknown to directly influence the conformation of CDRs, such as 48 and 49immediately preceding CDR2 of the HC, were also retained as murinesequence.

The amino acid sequence of the variable region for the initiallydesigned CDR-grafted TF8-5G9 HC, TF8HCDR1, is shown in SEQ NO.: 11.Murine residues were retained at framework positions 6, 17, 23, 24, 28,29, 30, 48, 49, 68, 71, 73, 78, 88 and 91. The CDR grafted HC variableregion was attached to a human IgG4 constant region.

The amino acid sequence of the variable region for the initiallydesigned CDR-grafted TF8-5G9 LC, TF8LCDR1, is shown in SEQ ID NO:12.Murine residues were retained at framework positions 39, 41, 46 and 105.The CDR-grafted LC variable region was attached to a human kappaconstant region.

The variable region for the CDR-grafted HC and LC described above wereeach assembled from 13 synthetic oligonucleotides where were synthesizedby Research Genetics, Inc., Huntsville, Ala. These oligonucleotidesranged in length from 42 to 80 bases, and encoded both variable regionstrands. When the 6 complementary oligonucleotide pairs were annealed,the overhangs generated were 17 to 24 bases in length. Theseoligonucleotide pairs were combined, annealed at their complementaryoverhangs, and ligated to give the final full length double-strandedvariable regions.

The HC variable region oligonucleotides were assembled into a 452 bpfragment which contains a 5′ EcoR1 site and a 3′ Sacl site. Thepolymerase chain reaction was used to amplify this fragment. Theresulting amplified DNA was purified on a 2% Nusieve, 1% Seakem agarosegel (FMC). The appropriate size band of DNA was excised and the DNA wasrecovered by the Geneclean (Bio 101) procedure. The fragment was thendigested with EcoR1 and Sac1, and purified again by the Genecleanmethod. This HC variable region fragment with EcoR1 and SacI ends wascloned into the EcoR1 and SacI sites of the pSport-1 vector (GIBCO-BRLLife Technologies, Gaithersburg, Md.). DNA from several clones wasisolated and sequenced to verify proper variable region assembly. Allclones had unexpected base changes. One clone with the fewest basechanges (two mismatches at bases 133 and 140) was selected to becorrected by site-directed mutagenesis according to Kunkel (1985) Proc.Natl. Acad. Sci. USA 82:488. Briefly, CJ236 (ung,dut-) competent cells(Invitrogen Corporation, San Diego, Calif.) were transformed with the psport vector containing the CDR-grafted HC variable region with the twobase mismatch. Single-stranded, uridine-incorporated DNA templates werepurified from phage following M13 helper phage (stratagene CloningSystems) infection of the transformed cells. Mutagenesis oligoscontaining the desired base changes were synthesized on an AppliedBiosystems Model 380B DNA synthesizer. The mutagenesis oligos wereannealed to the template DNA, and T7 DNA polymerase and T4 DNA Ligase(MutaGene InVitro Mutagenesis Kit, Bo-Rad Laboratories, Richmond,Calif.) were used to incorporate the oligo into a newly synthesized DNAstrano. DH5α competent cells (GIBCO-BRL Life Technologies) weretransformed with the double-stranded DNA. The originaluridine-incorporated strand is destroyed while the newly synthesizedstrand containing the mutagenesis oligo is replicated. Phagemid DNA wasprepared from the resulting mutagenesis clones and the variable regionswere sequence to identify the clones which had incorporated the desiredchanges. The corrected HC EcoRI/SacI variable region fragment wasexcised from the pSport vector, purified and ligated into the EcoRI/SacIsites of a pSG5 vector containing the human IgG4 constant region. Thisresulted in the generation of a full-length humanized TF8-5G9 HC gene,TF8HCDR1, in the pSG5 cos cell expression vector. The vector wasdesignated pSG5TF8HCDR1.

The CDR-grafted TF8-5G9 LC variable region was also amplified by the PCRfrom the assembled synthetic oligonucleotides into a 433 bp fragmentwhich contained a 5′ EcoRI site and a 3′ NarI site. This fragment waspurified as described above for the HC, digested with EcoRI and NarI andpurified by the Geneclean procedure. This fragment was cloned into theEcoRI and NarI sites of a pSG5 vector which contains the human kappaconstant region. This resulted in the generation of a full-lengthhumanized TF8-5G9 LC gene, TF8LCDR1, in the pSG5 cos cell expressionvector. Seven clones were sequenced, and one was found to have thedesired CDR. The vector was designated PSQ5TFBLCDR1.

EXAMPLE 4 Expression of the CDR-Grafted Heavy and Light Chain Genes inCOS Cells

The transient expression of antibody genes in COS-1 cells provides arapid and convenient system to test antibody gene expression andfunction. COS-1 cells were obtained from the American Type CultureCollection (CRL 1650) and cultured in Dulbecco's Modified Eagle Medium(DMEM, from GIBCO BRL Life Technologies) with 10% fetal calf serum. ThepSGSTF8HCDRI expression factor was cotransfected into COS cells with thepSGS chimeric LC expression vector using the DEAE-Dextran methodfollowed by DMSO shock as described by Lopata et al. (1984) NucleicAcids Res. 14:5707. After 4 days of culture, media was harvested fromthe wells and examined for antibody expression levels.

Antibody levels were determined by an ELISA based assembly assay. Plateswere coated with a goat anti-human Fc specific antibody. Variousdilutions of the COS cell supernatant containing secreted antibody wereadded, incubated for one hour, and washed. A horseradishperoxidase-linked goat anti-human kappa chain antibody was added,incubated for one hour at room temperature, and washed. Substrate forthe horseradish peroxidase was added for detection. Antibody levels inthe COS cell media were found to be nearly undetectable for theTF8HCDR1×chimeric LC. Upon closer examination of the TF8HCDR1 variableregion sequence, it was found that an unexpected base change, which hadoccurred during the site-directed mutagenesis process described inExample 3, introduced a stop codon into framework 4 of the TFBHCDRIgene. This substitution was corrected by site-directed mutagenesis asdescribed above. Thorough sequencing of the variable region confirmedthat the correction was made with no additional changes introduced. Upontransfection of this corrected TF8HCDRI gene with the chimeric LC,reasonable expression levels were obtained.

COS cells which had been co-transfected with the CDR-grafted LCexpression vector, pSGTF8LCDR1, and either the chimeric HC or TF8HCDR1,produced antibody at reasonable levels. Antibody levels in cas cellsupernatants ranged from 0.5 μg to 10.0 μg per ml.

EXAMPLE 5 Binding of the CDR-Grafted TF8-5G9 to Tissue Factor

An ELISA was used to determine the ability of the CDR-grafted TF8-5G9antibody, TF8HCDR1×TF8LCDR1, to bind to tissue factor. Tissue factor wasimmobilized on a microtiter plate. The test COS cell supernatant,containing the CDR-grafted antibody, was added to the well, incubatedfor one hour at room temperature. Following three washes with PBS/Tween,a goat anti-human kappa chain polyclonal antibody conjugated tohorseradish peroxidase was added, incubated for one hour at roomtemperature and washed. Substrate for the horseradish peroxidase wasadded for detection. The positive control was the TF8-5G9 chimericantibody. The CDR-grafted TF8-5G9 antibody was able to bind to tissuefactor to a degree comparable to the chimeric TF8-5G9 antibody (FIG. 3,solid symbols).

The ability of the humanized antibody to compete with murine TF8-5G9 forbinding to tissue factor was also examined. Varying amounts of COS cellsupernatant containing the test CDR-grafted antibody and a fixed amountof murine TF8-5G9 were added simultaneously to wells coated with tissuefactor. Binding was allowed to occur for one hour at room temperature.The wells were washed three times with PES/Tween. A goat anti-humankappa chain antibody conjugated to horseradish peroxidase was added,incubated for one hour at room temperature and washed. Substrate for thehorseradish peroxidase was added for detection. The positive antibodycompeted as well as the chimeric antibody with murine TF8-5G9 forbinding to TF.

These data indicate that the initially designed CDR-grafted antibody,TF8HCDR1×TF8LCDR1, was approximately as active as the chimeric TF8-5G9in binding to TF and competing with the murine antibody for binding toTF.

EXAMPLE 6 Construction and Characterization of Additional CDR-GraftedHeavy Chains

Upon examination of the molecular structure of murine TF8-5G9, frameworkresidues at positions 27, 68, 73 and 78 were found to lie on theantibody surface and had no discernible contact with the CDRs. Theseframework residues were of murine sequence in TF8HCDR1 but were changedto the human KOL sequence in various combinations to generate a seriesof CDR-grafted heavy chains with framework residue variations. Thechanges were made by the process of site-directed mutagenesis asdescribed in Example 3. Each CDR-grafted heavy chain version wasexpressed in COS cells in combination with the CDR-grafted LC, TF8LCDR1,and tested for its ability to bind TF and compete with murine TF8-5G9for binding. Every version of the CDR-grafted heavy chain in combinationwith TF8LCDR1 was shown to bind TF with an affinity comparable tochimeric TF8-5G9. Every CDR grafted HC in combination with TF8LCDR1 wasable to compete with murine TF8-5G9 for binding to TF to a degreecomparable to the chimeric antibody.

Changes in sequence from murine to human for HC framework positions 6,7, 68, 73 and 78 did not adversely affect the antigen binding ability ofthe antibody. The CDR-grafted HC version which had human sequence at allof these positions, and thus was the most humanized HC, was TF8HCDR20.

The complete sequence of the TF8HCDR20 gene was determined. The DNAsequence is shown as a 2360 bp EcoRI/BamHI insert with proteintranslation in the pEe6TF8HCDR20 expression vector in FIG. 4 and SEQ IDNO: 15

The essential regions of the gene are as follows:

Nucleotide # Region 1-6 5′ EcoRI restriction site  7-15 Kozak sequence16-72 Start codon and leader sequence  73-423 CDR-grafted variableregion 424-717 Human IgG4CH1 domain  718-1110 Human IgG4 intron 21111-1146 Human IgG4 hinge 1147-1267 Human IgG4 intron 3 1268-1594 HumanIgG4 CH2 domain 1595-1691 Human IgG4 intron 4 1692-2012 Human IgG4 CH3domain 2013-2354 3′ untranslated region 2355-2360 3′ BamHI end splicedto Bc1I site of the expression vector

EXAMPLE 7 Construction and Characterization of Additional CDR-GraftedLight Chains

The initially designed CDR-grafted LC, TF8LCDR1, contained fourframework residues from the murine TF8-5G9 sequence. At two of thesepositions, 39 and 105, the human REI framework sequence is unique toREI; however, the murine TF8-5G9 LC sequence is in agreement with thehuman consensus sequence. The other two murine framework residues, trp41and thr46, are unique to TF8-5G9. Several versions of the CDR-grafted LCwere generated in which the sequence at these four positions werechanged from the murine to the human REI in various combinations. Thesechanges were made by site-directed mutagenesis. Each version of theCDR-grafted LC was expressed in cos cells in combination with theCDR-grafted HC, TF8HCDR20, and tested for ability to bind tissue factorand compete with murine TF8-5G9 for binding. Every version of theCDR-grafted LC, in combination with TF8HCDR20, was shown to bind TF withan affinity comparable to TF8-5G9. Also every CDR-grafted LC version, incombination with TF8HCDR20, was able to compete with murine TF8-5G9 forbinding to TF in a manner comparable to the chimeric TF8-5G9 control.

Changes in sequence from murine to human for LC framework positions 39,41, 46 and 105 did not adversely effect the ability of the antibody torecognize antigen. The CDR-grafted LC of choice was TFBLCDR3, wheremurine TF8-5G9 sequence was used at positions 39 and 105 because theseare in agreement with the human consensus sequence. The preferredCDR-grafted TF8-5G9 antibody is TF8HCDR20×TF8LCDR3.

The complete sequence of the TF8LCDR3 gene was determined and is shownas a 759 bp EcoR1-BamH1 insert with protein translation in thepEe12TF8LCDR3 expression vector in FIG. 5 and SEQ ID NO: 17.

The essential regions of the gene are as follows:

Nucleotide # Region 1-5 5′ EcoR1 restriction site 6-8 Kozak sequence 9-68 Start codon and leader sequence  69-392 CDR-grafted variableregion 393-710 Human kappa constant region 711-753 3′ untranslatedregion 754-759 3′ BamH1 end spliced to Bc11 site of the expressionvector

EXAMPLE 8 CDR-Grafted TF8-5G9 Antibody TF8HCDR20×TF8LCDR3 Inhibits HumanTissue Factor

The binding of the CDR-grafted TF8-5G9 antibody, TF8HCDR20×TF8LCDR3, toTF was assessed as described in Example 5 and was found to be comparableto that of the chimeric TF8-5G9 as illustrated in FIG. 6. The ability ofthe CDR-grafted TF8-5G9 to compete with the murine antibody for bindingto TF is comparable to that of the chimeric TF8-5G9 as shown in FIG. 7.

An in vitro assay was used to measure the level of inhibition of factorX activation by the CDR-grafted TF8-5G9 antibody. In this assay, TFforms an active proteolytic complex with factor VII. This complex thenconverts factor X to factor Xa by proteolysis. The activated Xaenzymatically cleaves a substrate, Spectrozyme FXa, which releases achromogen. The level of chromogen, as detected by optical density, is anindication of factor X activation due to TF-factor VIIa activity.

The following reaction mixtures were prepared in 12×75 mm borosilicateglass tubes.

-   -   25 μl TBS (50 mM Tris, pH 7.4, 150 mM NaC1)    -   15 μl 20 mM CaCl₂/1% bovine serum albumin (BSA)    -   20 μl human placental tissue factor solution (prepared by        reconstituting one vial of Thromborel S, Curtin Matheson        Scientific #269-338 with 4.0 ml dH₂O and diluting 1:10 in TBS)    -   30 μl Factor VII (Enzyme Research Labs #HFVII 1007 at 237.66        ng/ml in TBS)    -   30 μl TBS or TF8-5G9 or TF8MCDR20×TF8LCDR3 at 1.18 μg/ml or as        indicated in FIG. 8

The reaction mixtures were incubated at 37° C. for ten minutes beforethe addition of Factor X. (In some cases the reaction mixture waspreincubated for five minutes before addition of Factor VII or antibody,followed by a ten-minute incubation before addition of Factor X.) Thirtyμ1 of Factor X solution (Enzyme Research Labs, DHFX 330, 247.38 μg/mlTBS) was added and the mixture was incubated at 37 C. for three minutes.Factor X activation was terminated by pipetting 40 μg of reactionmixture into 160 μl of stop buffer (50 mM Tris, pH 7.4, 100 mM EDTA, 150mM NaCI) in 96 well microtiter plates. Each tube of reaction mixture waspipetted into three microtiter wells. Fifty μI of Spectrozyme FXasubstrate (American Diagnostica #222, 1 μM/ml TBS) was added to eachwell 0D₄₀₅ was read on a Molecular Devices kinetic plate reader withreadings taken every twenty seconds for 10 minutes. Factor X activitywas recorded as mOD/minute, and enzyme velocities over the linearportion of the reaction curve were compared to determine inhibition offactor X activation by the anti TF antibodies.

As shown in FIG. 8, the CDR-grafted TF8-5G9 antibody is approximately aseffective as the murine TF8-5G9 in inhibiting factor X activation. Thisindicates that the CDR-grafted TF8-5G9 is functionally active.

EXAMPLE 9 Construction of the CDR-Grafted Heavy and Light Chain MyelomaExpression Vectors

For the purpose of establishing a permanent CDR-graftedantibody-producing cell line, the TF8HCDR20 and TF8LCDR3 genes weresubcloned into myeloma cell expression vectors. The heavy chainTF8HCDR20 was subcloned into the EcoRI and Bc1I sites of thepEe6hCMVBg1II myeloma expression vector described by Stephens et al.(1989) Nucleic Acids Res. 17:7110 to produce pEe6TF8HCDR20. The lightchain TF8LCDR3 was subcloned into the EcoTI and Bc1I sites of the pEe12myeloma expression vector to produce pEe12TF8LCDR3. The heavy and lightchain expression vectors are illustrated in FIGS. 9 and 10,respectively. In both vectors antibody gene transcription was driven bythe human cytomegalovirus (hCMV) promoter-enhancer, which lies directly5′ to the multiple cloning site. The polyadenylation signal sequencelies 3′ to the multiple cloning site and signals the termination oftranscription. Each vector contains the B-lactamase gene to allow forampicillin selection in E. coli. The pEe12 vector contains a glutaminesynthetase cDNA gene under the transcriptional control of the SV40 earlypromoter. Glutamine synthetase allows for myeloma cell transfectants tobe selected in glutamine-free media. Myeloma cells are devoid ofglutamine synthetase activity and are dependent on a supply of glutaminein the culture media. Cells which have been transfected with the pEe12vector, containing the glutamine synthetase gene, are able to synthesizeglutamine from glutamate and can survive in the absence of glutamine.

The pEe6TF8HCDR20 expression vector is a 7073 bp plasmid whose DNAsequence is shown in FIG. 4 and SEQ ID NO:15. The coding regions of theTF8HCDR20 gene are translated. The essential regions of this vector aredescribed below:

-   -   1 Nucleotides #1-2360: The TF8HCDR20 CDR-grafted HC gene is        described in Example 6. The HC gene was inserted as an        EcoRI/BamHI fragment into the EcoRI/Bc1I sites of the        pEe6hCMVBgII vector.    -   2 Nucleotides #2361-2593: This region encodes the SV40 early        gene polyadenylation signal (SV40 nucleotides 2770-2537), which        acts as a transcriptional terminator. This fragment is flanked        by a 5′ Bell site and a 3′ BamHI site. The 3′ BamHI end of the        heavy chain gene was spliced to the 5′ Bc1I site of the        polyadenylation signal, thus eliminating both sites.    -   3 Nucleotides #2594-3848: This region is a BamHI-Bg1-I fragment        from pBR328 (nucleotides 375-2422) but with a deletion between        the SaI and AvaI sites (pBR328 nucleotides 651-1425) following        the addition of a SaIl linker to the AvaI site. This region        contains the Col E1 bacterial origin of replication.    -   4 Nucleotides #3849-4327: This is a Bg1I-XmnI fragment site from        the B-lactamase gene of pSP64 (promega Corporation, Madison,        Wis.). This gene provides ampicillin resistance to bacteria        transformed with this vector.    -   5 Nucleotides #4328-4885: This is an XmnIHindIII fragment of the        Co1E1 based plasmid pCT54 described by Emtage et al. (1983)        Proc. Natl. Acad. Sci. USA 80:3671. The HindIII site was        converted to a Bq1II site by the addition of a linker following        the addition of the hCMV promoter described below.    -   6 Nucleotides #4886-7022: These nucleotides encode the Pst-1m        fragment of human cytomeglovirus (hCMV) strain AD 169 described        by Greenway et al. (1982) Gene 18:355 containing the region        coding for the hCMV middle intermediate early promoter. This        Pst-1m fragment was cloned into the HindIII site of pEe6hCMV by        addition of oligonucleotides of the following sequence to either        end of the fragment:

5′ GTCACCGTCCTTGACACGA 3′ (SEQ ID NO:21) 3′ ACGTCAGTGGCAGGAACTGTGCTTCGA5′ (SEQ ID NO:22)The resulting 2100 bp fragment was inserted such that the promoterdirected transcription towards the EcoRI site of pEe6hCMV. Theoligonucleotide above served to recreate the complete 5′ untranslatedsequence of the hCMV-MIE gene the added irrelevant sequence at the very5′ end of the fragment. The HindIII site at the 5′ end was subsequentlyconverted to a Bg1II site by the addition of a further linker.

-   -   7 Nucleotides #7023-7073: The pSP64 polylinker with the BamHI        and SaI1 sites removed.

The pEe12TF8LCDR3 expression vector is a 7864 bp plasmid whose DNAsequence is shown in FIG. 5 and SEQ ID NO:17. The coding regions of theTF8LCDR3 gene are translated. The essential regions of this expressionvector are described below:

-   -   1 Nucleotides #1-759: The TF8LCDR3 CDR-grafted LC gene is        described in Example 7. The gene was inserted as an EcoRI/BamHI        fragment into the EcoRI/Bc1II sites of the pEe12 expression        vector.    -   2 Nucleotides #760-3284: These regions of pEe12 are identical to        the regions encoded by nucleotides 2361-4885 of the        pEe6TF8HCDR20 (regions #2-5).    -   3 Nucleotides #3285-5736: This region encodes the Chinese        hamster ovary glutamine synthetase cDNA under the        transcriptional control of the SV40 early promoter and followed        by the SV40 polyadenylation an splice signals from the pSV2.dhfr        vector described by Subramani et al. (1981) Mol. Cell. Biol.        1:854. The following describes the derivation of this region: A        1200 bpNaeI-PvuII fragment, containing a complete GS coding        sequence, was excised from the Chinese hamster ovary cDNA clone        AGS 1.1 described by Hayward et al. (1986) Nucleic Acid Res.        14:999. After addition of a HindIII linker to the NaeI site and        a Bg1II linker to the PvuII site (hence destroying the NaeI and        PvuII sites), the 1200 bp fragment was cloned in place of DHFR        sequences in pSV2.dhfr between the HindIII and Bg1II sites to        form pSV2.GS. The single remaining PvuII site in pSV2BamGS was        converted to a BamHIl site by addition of an oligonucleotide        linker to form pSV2BamGS. An EcoRI site in the GS cDNA was        destroyed by site directed mutagenesis without altering the        amino acid sequence in pSV2BamGS and the HindIII site was        destroyed by filling in with DNa polymerase I. The 2451 bp BamHI        fragment from this plasmid, containing the complete SV40-GS        hybrid transcription unit, was excised and inserted at the Bq1II        site of pEe6hCMV-Bg1II site of pEe6hCMV-Bg 1II such that        transcription from the sV40 early promoter proceeds towards the        hCMV promoter.    -   4 Nucleotides #5737-7864: This region is identical to the hCMV        promoter and pSP64 polylinker encoded by nucleotides 4886-7073        of the pEe6TF8HCDR20 vector described above (regions 6 and 7).

For the purpose of ensuring that both the pEe6TF8HCDR20 andpeE12TF8LCDR3 vectors co-transfected myeloma cells, the vectors werejoined in linear concatamers. Both the pEe6TF8HCDR20 and pEe12TF8LCDR3vectors were digested at the unique SaI1 site. The SaI1 linearizedpEe6TF8HCDR20 vector was phosphatased at its 5′ ends to prohibitligation of two pEe6TF8HCDR20 vectors onto each other. This phosphatasedHC vector was ligated in a 2:1 molar ratio to the Sal linearizedpEe12TF8LCDR3. The resulting concatamers were most likely of thefollowing composition:

This concatamerized DNA was extracted with phenol and chloroform, andprecipitated with ammonium acetate and ethanol. The DNA precipitate wasresuspended in distilled water to a concentration of 1 μg/μL and used totransfect myeloma cells.

EXAMPLE 10 Development of NSO Expression Cell Lines

Stably transformed cell lines expressing the humanized TF8-5G9 antibodywere prepared by transfecting CDR-grafted heavy and light chainexpression vectors into NSO mouse myeloma cells. Selection oftransfected cells was carried out using the dominant selectable markergene, glutamine synthetase (GS).

The NSO mouse myeloma cell line, obtained from Celltech, Ltd., is asubclone derived from NS-1 and does not express intracellular lightchains. These cells were cultured in Dulbecco's modified Eagle's medium(DMEM) with added glutamine and 10% fetal bovine serum (FBS). To preparefor transfection, the cells were harvested in mid-log phase of thegrowth cycle, centrifuged for 5 minutes, washed with phosphate bufferedsaline (PBS), centrifuged again, and the cell pellet was resuspended in2.2 mL of PSS. The final cell concentration was 2.18×107 mL. Cells weremaintained on ice during the entire procedure.

The DNA to be transfected (pEe12TF8LCDR3×pEe6TFBHCDR20) was prepared asa concatamer as described in Example 9. The DNA and NSO cells were addedto a 0.4 cm BioRad Gene Pulse cuvette in the following order:

-   -   40 μL (40 μg) DNA concatamer    -   320 μL double distilled water    -   40 μL 10×PBS    -   400 μL NSO cells (8.72×106 cells).

Transfection was performed by electroporation following a protocolprovided by Celltech, Ltd. In this procedure, the cells and DNA in PBSbuffer were exposed to a brief, high voltage pulse of electricitycausing transient micropores to form on the cell membrane. DNA transfertakes place through these openings. To prepare for electroporation, thesuspension of NSO cells and DNA was gently mixed and incubated on icefor 5 minutes. The cuvette was placed in a BioRad Gene Pulser and given2 consecutive electrical pulses at settings of 3 μF (capacitance) and1.5V (voltage). Following electroporation, the cuvette was returned tothe ice for 5 minutes. The suspension was then diluted in prewarmedgrowth medium and distributed into seven 96-well plates. Control platescontaining cells electroporated without DNA were also prepared at thesame time to measure the presence of spontaneous mutants. Plates wereplaced in a 37° C. incubator with 5 % CO2.

Glutamine synthetase, encoded by the GS gene, is an enzyme that convertsglutamate to glutamine. NSO cells require glutamine for growth due toinadequate levels of endogenous GS gene expression. In the DNAconcatamer, this gene is located on the pEe12TF8LCDR3 vector.Transfected cells which incorporate the GS gene becomeglutamine-independent. Cells not integrating the GS gene into theirgenome would remain glutamine dependent and would not survive inglutamine-free medium. Approximately 18 hours post electroporation, allplates were fed with glutamine-free selection medium and returned to theincubator until visible colonies appeared.

Approximately 3 weeks after transfection, distinct macroscopic colonieswere observed. These were screened for expression of the intacthumanized antibody using the assembly ELISA as described in Example 5.

Tissue culture supernatants from wells containing 1 colony were screenedat a 1: 10 dilution. Positive wells showing activity greater than the 25ng/mL standard were subcultured and expanded for further analysis.

For selection of high producers, antibody production was quantitatedafter a 96 hour growth period. Tissue culture flasks were seeded with2×10⁵ cells/mL in 10 mL of selection medium and incubated at 37° C., 5%CO₂ for 96 hours. At the end of that time period, an aliquot was takento determine cell concentration and antibody titer. Evaluation ofantibody production was calculated as μg/mL and pg/cell/96 hours. Thehighest producers from this transfection were:

Cell Line μg/mL pg/cell/96 hour 2B1 26.3 24.3 3E11 27.6 59.9 4G6 30.241.9

EXAMPLE 11 CDR-Grafted Antibody TF8HCDR20×TF8LCDR3 Inhibits TissueFactor in Vivo

CDR-grafted antibody TF8HCDR20×TF8LCDR3 was compared to murine antibodyTF8-5G9 for its ability to protect rats from experimentally induceddisseminated intravascular coagulation (DIC). In the DIC model, rats arechallenged with human thromboplastin (a crude tissue extract containingTF activity), resulting in fibrinogen consumption and death.Pretreatment of rats with antiTF antibody was demonstrated to protectrats from fibrinogen consumption and death as follows.

Human thromboplastin was prepared as described in U.S. Pat. No.5,223,427. Saline control or 30 μ/ml of TF8-5G9 or CDR-grafted antibodywas injected through the tail vein of rats, followed by injection ofthromboplastin equivalent to 200 ng of recombinant TF. Clotting timeswere determined at T=O and T=1 minute as a measure of fibrinogenconcentration. Clotting times are proportional to fibrinogenconcentration, with a 60 second clotting time corresponding to an 80%reduction in fibrinogen concentration. Clotting times of greater than 60seconds cannot be accurately measured and were recorded as 60 seconds.Survivability and clotting times for three representative studies areshown below.

Survivors CDR-grafted Study Controls TF8-5G9 Ab 1 0/8 5/8 6/8 2 0/8 4/77/8 3 0/8 8/8 3/7 Clotting Times Controls Study #1 Study #2 Study #3 T =O T = 1 T = O T = 1 T = O T = 1 16 >60 18 >60 19 >60 16 >60 18 >6021 >60 16 >60 18 >60 18 >60 17 >60 18 >60 19 >60 15 >60 16 >60 18   5416 >60 18 >60 18 >60 16 >60 17 >60 18 >60 16 >60 17 >60 18 >60 ClottingTimes Murine TF8-5G9 Study #1 Study #2 Study #3 T = O T = 1 T = O T = 1T = O T = 1 16 36 18 34 19 28 15 41 18 36 18 29 15 33 18 >60   19 29 1531 17 >60   18 29 15 >60   18 50 18 28 16 >60   17 34 19 40 16 33 17 3419 40 16 33 18 31 19 34 16 >60   19 >60   Clotting Times CDR-graftedTF8-5G9 Study #1 Study #2 Study #3 T = O T = 1 T = O T = 1 T = O T = 116 >60 17 >60   21 >60   16 >60 17 33 18 34 16 >60 18 32 17 >60   22  37 18 >60   20 35 16   32 17 32 17 58 15 >60 18 31 18 33 16 >60 17 3118 31 16 >60 16 32

Twenty-three of the twenty-four control rats had clotting times ofgreater than 60 seconds indicating that virtually all untreated ratswere consuming more than 80% of their fibrinogen. Both the COR-graftedand murine antibody treated rats had similar clotting times at oneminute of 44.5 and 40 seconds. Further, only six of the murine antibodytreated rats and nine of the COR-grafted antibody treated rats hadclotting times in excess of 60 seconds. Accordingly, both the murine andCDR-grafted antibodies were able to neutralize TF and thus protect ratsfrom fibrinogen consumption and death.

1. An isolated nucleic acid encoding the heavy chain of a CDR-graftedantibody capable of inhibiting human tissue factor, wherein thecomplementarity determining regions (CDRs) are obtained from a murinemonoclonal antibody against human tissue factor and the constant (C) andframework (FR) regions are obtained from one or more human antibodies,wherein said CDRs of the heavy chain have the amino acid sequences: CDR1DYYMH (SEQ ID NO: 5) CDR2 LIDPENGNTIYDPKFQG (SEQ ID NO: 6) CDR3 DNSYYFDY(SEQ ID NO: 7)

and said CDRs of the light chain have the amino acid sequences: CDR1KASQDIRKYLN (SEQ ID NO: 8) CDR2 YATSLAD (SEQ ID NO: 9) CDR3 LQHGESPYT(SEQ ID NO: 10)

wherein the heavy chain comprises residues obtained from the murinemonoclonal antibody at positions 23, 24, 28, 29, 30, 48, 49, 71, 88 and91 and the light chain comprises residues obtained from the murinemonoclonal antibody at positions 39 and 105, wherein the residues arenumbered according to the Kabat numbering system.
 2. An isolated nucleicacid encoding the light chain of the CDR-grafted antibody of claim
 1. 3.An isolated nucleic acid encoding a heavy chain variable region havingthe amino acid sequence of SEQ ID NO:
 13. 4. An isolated nucleic acidencoding a light chain variable region having the amino acid sequence ofSEQ ID NO:
 14. 5. The nucleic acid of claim 3 having the sequence ofnucleotides 1-2360 of SEQ ID NO:
 15. 6. The nucleic acid of claim 4having the sequence of nucleotides 1-758 of SEQ ID NO: 20.